The development of new systems for the delivery or release of active principles has as a first object the controlled delivery of an active agent, especially a pharmacological agent, to its site of action at a therapeutically optimal rate and dosage (J. Kreuter, 1994). The improvement of the therapeutic index can be obtained by modulation of the distribution of the active principle in the organism. The association of the active principle with the delivery system makes possible its specific delivery to the site of action or of its controlled release after targeting the site of action. The reduction in the amount of active principle in the compartments in which its presence is not desirable makes it possible to increase the efficacy of the active principle, to reduce its toxic side effects and even to modify or restore its activity.
One of the major issues of delivery is its application to molecular biology and, most particularly, its application to active principles such as deoxyribonucleic acids (DNAs), oligodeoxynucleotides (ODNs), peptides, proteins—all negatively charged. The molecular biology bases in genetic diseases makes it possible to modulate or replace a dysfunctional gene. The development of gene therapy as routine in clinical practice is dependent on the possibility of repeated administration via the systemic route, the capacity to reach a target and to effectively transfect cells in vivo (Felgner, 1990; Kabanov and Alakhov, 1993; Crook, 1995; Douglas and Curiel, 1995; Lasic and Templeton, 1996) as well as the possibility of fabricating vectors which will be capable of being adapted to an industrial scale. The techniques of in vitro transfection used to date, such as electroporation and co-precipitation of DNA with calcium phosphate, are approaches the application of which in vivo would appear to be difficult (Fynan et al., 1993).
The systems for in vivo transfection take into account the natural negative charge of DNAs and ODNs. These are either viral systems (Morgan and Anderson, 1993) or nonviral constructions such as cationic lipids (Ledley, 1994; Zelphati and Szoka, 1996). The viral vectors are very effective in terms of in vitro transfection, but they have limitations in vivo because of their immunogenicity (Wilson et al., 1990; Douglas and Curiel, 1995; Verma and Somia, 1997). Among the nonviral vectors, the cationic lipids such as Transfectam (Behr et al., 1989) have good transfection properties, but their application in vivo is limited by their toxicity, the activation of the complement and their notable tropism for the liver and the lungs.
Since the studies carried out with poly(1-lysine) at the end of the 1980s (Wu and Wu, 1987), many cationic polymers have been studied as nonviral transfection vectors. These vectors include polyethyleneimine (Boussif et al., 1995; Remy et al., 1998), polybrene (Mumper et al., 1996) and the dendrimers of poly(amidoamine) (PAMAM) (Tang et al., 1996).
Until now, the efficacy of transfection with polymers is relatively low and many of these cationic polymers have exhibited a relative toxicity with low potentials of repeated administration via the intravenous route. The polymers used directly with the active principles notably DNA are limited by their transfection efficacy and consume a large amount of active principle and thus require the use of a large amount of polymer, which is itself toxic. The colloidal systems used for the delivery notably of genes in vivo use smaller amounts of polymer and active principle.
The colloidal delivery systems of active principles comprise the liposomes, microemulsions, nanocapsules, nanospheres, microparticles and nanoparticles. Nanoparticles have advantages of targeting, modulation of distribution and flexibility of formulation and have a polymer structure which can be designed and implemented in a manner adapted to the goal. They have been found to be promising for obtaining a better therapeutic index in the sense defined above because of their aptitude to ensure a controlled release, a specific delivery to the site of action or targeted delivery, enabling both an augmentation of the efficacy and a reduction in the toxic side effects at the level of the other organs.
Among these, the poly(alkyl cyanoacrylates) described in EP-B-0 007 895 (U.S. Pat. No. 4,329,332 and U.S. Pat. No. 4,489,055) and EP-B-0 064 967 (U.S. Pat. No. 4,913,908) are particularly interesting because their bioerosion takes place rapidly in relation to other biodegradable polymers and unfolds over durations compatible with therapeutic and diagnostic applications. The nanoparticles are colloidal vectors the diameter of which ranges between 10 nm and 1000 nm. These particles are formed by macromolecules in which the biologically active substance is trapped, encapsulated or adsorbed at the surface. Nanoparticles or nanospheres are described by Birrenbach and Speiser (1976) in terms of nanopellets and nanocapsules and qualified by Kreuter and Speiser (1976) as adjuvants and delivery systems of active substances by Kreuter (1983).
With the goal of increasing the stability of the oligonucleotides, of increasing their penetration into the cells and avoiding nonspecific distribution, the use of particular vectors, like nanoparticles, is considered to be one of the most promising approaches. However, their use has been limited by the toxicity of the substance used to affix the active principle to the nanoparticle.
The polymer nanoparticles that have been subjected to the greatest amount of research are the polyisohexylcyanoacrylates (PIHCA). However, as nanoparticles having a negative surface charge, a cationic copolymer (diethylaminoethyl (DEAE) dextran) or a cationic hydrophobic detergent (cetyl trimethyl ammonium bromide (CTAB)) were combined with polyalkylcyanoacrylates (PACA) to facilitate association of ODNs by formation of ion pairs on the nanoparticles. Thus, the ODNs were effectively associated with the PACA nanoparticles containing a hydrophobic cation such as CTAB (cetyl trimethyl ammonium bromide, Chavany et al., 1992). Since CTAB has toxicity problems, Zobel et al., 1987 replaced the CTAB with DEAE dextran which was introduced into the polymerization medium prior to formation of the nanospheres. DEAE dextran was also found to be toxic.
A desirable nanoparticle vector for delivery of active principles such as DNAs, ODNs, peptides and proteins should be:
capable of forming a complex with the molecule of interest taking into account its physicochemical characteristics and the substance selected for affixing the active principle must be adapted to the charge and the toxicity data,
capable of protecting the active principle from degradation during its transport in the circulating blood,
biocompatible (nontoxic, nonimmunogenic and preferably biodegradable),
capable of delivering the active principle to the level of the target tissue in sufficient quantity, and
capable of targeting specifically a cell type.